Project Summary Metastasis accounts for nearly all breast cancer-related deaths. Bone is the organ most frequently affected by breast cancer. In the clinically significant stage, bone metastasis is driven by a vicious cycle between cancer cells and osteoclasts (bone-resorbing cells). Our knowledge of this vicious cycle has vastly increased in recent years, and therapies targeting osteoclasts can often significantly delay the progression of disease. However, bone metastases still remain incurable. On the other hand, there is often a latency of years to decades before bone metastases become clinically detectable, suggesting that residual cancer cells can exist in bone or bone marrow for a protracted period of time without activating osteoclasts. We set out to discover osteoclast- independent mechanisms in early-stage bone colonization before the onset of the vicious cycle. Our prelimi- nary data demonstrated that osteoblasts (bone-making cells) and their precursor cells constitute the microenvi- ronment niche of microscopic bone metastases. The direct cell-cell contact between cancer cells and the os- teoblastic niche is crucial for their proliferation. Further studies indicated that the re-activation of the mTOR pathway is a hallmark of bone metastasis initiation. We also obtained preliminary evidence suggesting that the activation of mTOR is mediated by the formation of adhesion junctions (AJs) between cancer cells and niche cells. Based on these findings, we hypothesize that the osteoblastic niche facilitates bone metastasis progres- sion of breast cancer from single cells to multi-cell micrometastases by augmenting the activity of the mTOR pathway, possibly through signaling downstream of AJ complexes. To test this hypothesis, we will pursue two specific aims: 1) to determine the mechanism mediating the crosstalk between cancer cells and the osteo- blastic niche, which entails direct cell-cell contact and leads to the activation of mTOR signaling, and 2) to iden- tify the downstream effectors of mTOR that drive metastasis initiation. Our work is innovative and feasible be- cause it employs a novel technique that selectively delivers cancer cells into hind limb bones via the circulation. This approach enables swift inspection and robust quantification of bone micrometastases at a single-cell reso- lution, yet avoids caveats of other conventional approaches. Application of this technique to several cancer models resulted in indolent or dormant bone metastases that mimic human diseases. We will use this ap- proach for xenograft and syngeneic transplantation of human and mouse cancer cells, respectively, and inves- tigate the roles of AJs, the mTOR complexes, and their related signaling molecules in bone metastasis initia- tion. In addition, we also invented a 3D co-culture system that faithfully recapitulates many features of cancer- niche interaction, which will facilitate the dissection of molecular mechanisms and accelerate our examination of candidate mediators. The fulfillment of these aims will enable the design of targeted therapies to suppress or eradicate latent tumor cells, and reduce the incidence of overt bone metastasis-related symptoms and mortality.